The baculovirus expression vector system (BEVS) has been successfully utilized to produce thousands of proteins for use as vaccines and therapeutics as well as for studies of protein structure and function. One limitation of BEVS is the propensity of baculoviruses to accumulate transposon insertions into the fp25k gene, leading to the few polyhedra (FP) phenotype. This mutation shifts the balance of virus production from occlusion-derived viruses, which are not infectious in tissue culture, to budded viruses (BV), which are the form of virus that is used in baculovirus expression. Higher levels of BV would be advantageous for BEVS users but FP mutants are also deficient in transcription from the polyhedrin promoter that drives expression of target genes. Baculoviruses also rapidly accumulate defective interfering particles (DIP), which are often linked to a sharp decrease in target gene expression, due to deletion of the target gene and/or the viral genes needed for its expression. One factor promoting DIP formation is transposition into fp25k. The goal of this proposal is to limit deleterious effects of transposition into fp25k, while taking advantage of the fact that elimination of the FP25K activity significantly increases BV production. During Phase I, two strategies - an inducible construct for controlling fp25k and an fp25k deletion mutant coupled with a cell line constitutively expressing FP25K - were explored for regulating FP25K expression. Both strategies sought to produce high titer virus during the amplification stage of baculovirus infection and enable a switch to high level transcription from the polyhedrin promoter during the recombinant protein expression phase. Results document achievement of Phase I objectives with deletion of fp25k from the virus and complementary expression from an engineered cell line enabling the predicted control of budded virus and recombinant protein production. However, the inducible construct did not provide sufficient FP25K control due to the toxicity of the heavy metal inducer to insect cells. Therefore, in Phase II, we will (1) develop an improved inducible construct with tighter transcriptional regulation, using an ecdysone receptor-based inducible promoter; (2) validate the beneficial effects of FP25K regulation using viruses that express intracellular and secreted yellow fluorescent protein (YFP) for BEVS and BacMam (mammalian expression) technology; (3) develop user friendly fp25k mutant backbones for simplified BEVS and BacMam cloning and demonstrate their utility with several medically relevant transgenes; and (4) test the fp25k expression system in the context of vankyrin-enhanced BEVS (VE-BEVSTM) technology. ParaTechs' VE-BEVS products delay death and lysis of baculovirus-infected cells, thereby boosting target protein expression up to 20 fold. Many BEVS users would want to incorporate both vankyrin and FP25K technologies; therefore, it is important to determine whether they are compatible. Taken together, completion of Phase II objectives will enable users of the BEVS to control and optimize production of BV and recombinant protein expression.